Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP(8) Levels

The HCT116 cell line, which has a pseudo-diploid karotype, is a popular model in the fields of cancer cell biology, intestinal immunity, and inflammation. In the current study, we describe two batches of diverged HCT116 cells, which we designate as HCT116(NIH) and HCT116(UCL). Using both gel electrophoresis and HPLC, we show that HCT116(UCL) cells contain 6-fold higher levels of InsP(8) than HCT116(NIH) cells. This observation is significant because InsP(8) is one of a group of molecules collectively known as ‘inositol pyrophosphates’ (PP-InsPs)—highly ‘energetic’ and conserved regulators of cellular and organismal metabolism. Variability in the cellular levels of InsP(8) within divergent HCT116 cell lines could have impacted the phenotypic data obtained in previous studies. This difference in InsP(8) levels is more remarkable for being specific; levels of other inositol phosphates, and notably InsP(6) and 5-InsP(7), are very similar in both HCT116(NIH) and HCT116(UCL) lines. We also developed a new HPLC procedure to record 1-InsP(7) levels directly (for the first time in any mammalian cell line); 1-InsP(7) comprised <2% of total InsP(7) in HCT116(NIH) and HCT116(UCL) lines. The elevated levels of InsP(8) in the HCT116(UCL) lines were not due to an increase in expression of the PP-InsP kinases (IP6Ks and PPIP5Ks), nor to a decrease in the capacity to dephosphorylate InsP(8). We discuss how the divergent PP-InsP profiles of the newly-designated HCT116(NIH) and HCT116(UCL) lines should be considered an important research opportunity: future studies using these two lines may uncover new features that regulate InsP(8) turnover, and may also yield new directions for studying InsP(8) function.